Virus Breakthrough Research Articles

Mucosal nirsevimab levels in respiratory syncytial virus breakthrough bronchiolitis

Mucosal nirsevimab levels in respiratory syncytial virus breakthrough bronchiolitis

Molecular surveillance in the nirsevimab era: lessons from respiratory syncytial virus breakthrough infections

Molecular surveillance in the nirsevimab era: lessons from respiratory syncytial virus breakthrough infections

Application and Significance of SIRVB Model in Analyzing COVID-19 Dynamics.

In the summer of 2024, COVID-19 positive cases spiked in many countries, but it is no longer a deadly pandemic thanks to global herd immunity to the SARS-CoV-2 viruses. In our physical chemistry lab in spring 2024, students practice kinetic models, SIR (Susceptible, Infected, and Recovered) and SIRV (Susceptible, Infected, Recovered, Vaccinated) using COVID-19 positive cases and vaccination data from World Health Organization (WHO). In this report, we further introduce virus breakthrough to the existing model updating it the SIRVB (Susceptible, Infectious, Recovered, Vaccinated, Breakthrough) model. We believe this is the simplest model possible to explain the COVID-19 kinetics in all countries in the past four years. Parameters obtained from such practice correlate with many indices of different countries. These models and parameters have significant value to researchers and policymakers in predicting the stages of future outbreaks of infectious diseases.

Pilot evaluation of the potential log removal credit using chemical markers for reverse osmosis and nanofiltration

Reverse osmosis (RO) and nanofiltration (NF) are widely applied membrane treatment technologies due to their ability to effectively remove particulates, dissolved compounds, bacteria, and viruses. However, in potable reuse applications, RO is given minimal log removal value (LRV) credit of 1–2 for pathogens due to the inability to demonstrate higher LRV performance daily. Therefore, systematic single-element membrane pilot tests (3 different RO and 1 NF membranes) were performed to assess the potential LRVs (> 4) for three chemical markers (i.e., sulfate, sucralose, and uranine) that could potentially enable membrane-based potable reuse systems to achieve higher credit. Lead- and tail-element tests, as well as new and chlorine-oxidized membranes, were tested to assess the effect of membrane location and damage on the marker LRVs. Undamaged and damaged membranes demonstrated LRVs of >5 for MS2 bacteriophage with no statistically significant differences observed for any of the 4 membranes tested regardless of membrane placement (i.e., lead vs. tail) or condition (i.e., new vs. oxidized). Statistical analysis by optimal cluster assignments for each membrane indicated there were little differences in effective LRVs between the three chemical markers. Though the LRVs could not achieve >4, likely due to diffusion limitations, they were > 2 for the three chemical markers that ranged from 2.7 to 3.6 across non-damaged lead- and tail-element tests for all three RO membranes. Uranine, while achieving LRVs of 2.9–3.6 for RO and 2.2–2.3 for NF for non-damaged lead- and tail-element tests, was deemed less preferable due to its high dose requirement and difficult chemical handling. Sulfate and sucralose proved to be effective as well but preferable with non-damaged, lead- and tail-element test LRVs of 2.7–3.5 for RO and 1.4–2.3 for NF. Damaged-membrane testing demonstrated the three conservative chemical markers were sensitive to the chlorine-oxidized membrane compromise, more so for RO than NF, before any virus breakthrough occurred.

Virus breakthrough behavior during virus filtration: Effects of virus/protein concentration and operating conditions

Virus filtration in biopharmaceutical downstream process ensures viral safety with removal efficiency greater than 99.99%. Despite its robust performance, it has been reported that viruses can escape through the membrane under certain conditions. This study aimed to investigate virus breakthrough using Viresolve® Pro membrane with PP7 bacteriophage under high bacteriophage titer, high protein concentration, flow interruption, and low-flux operation. The results show high virus removal performance (LRV >6.8) up to 109 PFU/mL for 200 L/m2, with the first phage breakthrough observation over 1012 PFU/m2 of phage challenge in the membrane. As the protein concentration increased in the coexistence condition, a greater number of phages passed through the membrane. During the post-buffer flush and the operation at low filtrate flux, diffusion plays an important role in the escape of phages, which were detected as high as 1012 PFU/m2, but as low as 109 PFU/m2. These results suggest that the virus-retentive membrane may become susceptible to undesired virus breakthrough when exposed to phage challenges of > 1012 PFU/mL, providing important key factors to ensure viral safety during the downstream process of biopharmaceutical production.

Effect of protein fouling on filtrate flux and virus breakthrough behaviors during virus filtration process.

Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log10 ). However, it is still constrained by protein fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.

Prioritizing interventions for preventing COVID-19 outbreaks in military basic training.

Like other congregate living settings, military basic training has been subject to outbreaks of COVID-19. We sought to identify improved strategies for preventing outbreaks in this setting using an agent-based model of a hypothetical cohort of trainees on a U.S. Army post. Our analysis revealed unique aspects of basic training that require customized approaches to outbreak prevention, which draws attention to the possibility that customized approaches may be necessary in other settings, too. In particular, we showed that introductions by trainers and support staff may be a major vulnerability, given that those individuals remain at risk of community exposure throughout the training period. We also found that increased testing of trainees upon arrival could actually increase the risk of outbreaks, given the potential for false-positive test results to lead to susceptible individuals becoming infected in group isolation and seeding outbreaks in training units upon release. Until an effective transmission-blocking vaccine is adopted at high coverage by individuals involved with basic training, need will persist for non-pharmaceutical interventions to prevent outbreaks in military basic training. Ongoing uncertainties about virus variants and breakthrough infections necessitate continued vigilance in this setting, even as vaccination coverage increases.

HOPE in action: A prospective multicenter pilot study of liver transplantation from donors with HIV to recipients with HIV

Liver transplantation (LT) from donors-with-HIV to recipients-with-HIV (HIV D+/R+) is permitted under the HOPE Act. There are only three international single-casereports of HIV D+/R+ LT, each with limited follow-up. We performed a prospective multicenter pilot study comparing HIV D+/R+ to donors-without-HIV to recipients-with-HIV (HIV D-/R+) LT. We quantified patient survival, graft survival, rejection, serious adverse events (SAEs), human immunodeficiency virus (HIV) breakthrough, infections, and malignancies, using Cox and negative binomial regression with inverse probability of treatment weighting. Between March 2016-July 2019, there were 45 LTs (8 simultaneous liver-kidney) at 9 centers: 24 HIV D+/R+, 21 HIV D-/R+ (10 D- were false-positive). The median follow-up time was 23months. Median recipient CD4 was 287cells/µL with 100% on antiretroviral therapy; 56% were hepatitis C virus (HCV)-seropositive, 13% HCV-viremic. Weighted 1-year survival was 83.3% versus 100.0% in D+ versus D- groups (p=.04). There were no differences in one-year graft survival (96.0% vs. 100.0%), rejection (10.8% vs. 18.2%), HIV breakthrough (8% vs. 10%), or SAEs (all p>.05). HIV D+/R+ had more opportunistic infections, infectious hospitalizations, and cancer. In this multicenter pilot study of HIV D+/R+ LT, patient and graft survival were better than historical cohorts, however, a potential increase in infections and cancer merits further investigation.

Particle-based analysis elucidates the real retention capacities of virus filters and enables optimal virus clearance study design with evaluation systems of diverse virological characteristics.

In virus clearance study (VCS) design, the amount of virus loaded onto the virus filters (VF) must be carefully controlled. A large amount of virus is required to demonstrate sufficient virus removal capability; however, too high a viral load causes virus breakthrough and reduces log reduction values. We have seen marked variation in the virus removal performance for VFs even with identical VCS design. Understanding how identical virus infectivity, materials and operating conditions can yield such different results is key to optimizing VCS design. The present study developed a particle number‐based method for VCS and investigated the effects on VF performance of discrepancies between apparent virus amount and total particle number of minute virus of mice. Co‐spiking of empty and genome‐containing particles resulted in a decrease in the virus removal performance proportional to the co‐spike ratio. This suggests that empty particles are captured in the same way as genome‐containing particles, competing for retention capacity. In addition, between virus titration methods with about 2.0 Log10 difference in particle‐to‐infectivity ratios, there was a 20‐fold decrease in virus retention capacity limiting the throughput that maintains the required LRV (e.g., 4.0), calculated using infectivity titers. These findings suggest that ignoring virus particle number in VCS design can cause virus overloading and accelerate filter breakthrough. This article asserts the importance of focusing on virus particle number and discusses optimization of VCS design that is unaffected by virological characteristics of evaluation systems and adequately reflect the VF retention capacity.

Mechanistic insights into flow-dependent virus retention in different nanofilter membranes

Virus filtration is a downstream unit operation in the manufacturing of biotherapeutics to remove potential viral contaminations based on size exclusion. While even very small viruses are effectively retained under normal flow conditions, process interruptions can compromise the virus-reduction capacity of a filter. Yet, direct insights into the underlying flow-dependent retention and breakthrough mechanisms of relevant parvoviruses are still lacking. To study the retention of parvoviruses inside the polymeric structures of four commonly used filter types, minute virus of mice (MVM) was fluorescently labeled and visualized in membrane cross-sections post-filtration by laser scanning microscopy. The virus retention profiles revealed a membrane structure-specific accumulation of viral particles at a distinct depth in the separation-active layers. Pressure release experiments showed that flow interruption-induced virus breakthrough is associated with the mobilization and deeper migration of viruses into denser membrane layers. Moreover, we discovered that local clusters of breakthrough foci in a particular filter type are responsible for the substantial transmission of viruses to the filtrate. Taken together, the membrane-specific phenomena visualized herein contribute to a better understanding of the underlying virus retention mechanisms and provide cues for a specific optimization of virus filtration processes.

The effects of flux on the clearance of minute virus of mice during constant flux virus filtration.

Constant flux virus filtration experiments were conducted to evaluate minute virus of mice retention behavior of four commercial virus filters for continuous bioprocessing applications. Fluxes chosen were guided by the Peclet number and the processing logistics as well as based on the filter characteristics. At the low flux condition of 5 (LMH) when diffusive force dominates, a significant breakthrough was observed for all the filtrate fractions for the filtration of a low fouling monoclonal antibody for three of the four filters. When both diffusive and convective forces are equally important at 40 LMH, virus breakthrough in buffer chase was observed only in one of the four filters investigated. When convective force dominates at 60 LMH or above, a high degree of virus clearance was observed for all three parvovirus filters investigated. Our work shed light on virus clearance during constant flux virus filtration for future continuous biomanufacturing.

Development and Evaluation of a Machine Learning-Based In-Hospital COVID-19 Disease Outcome Predictor (CODOP): A Multicontinental Retrospective Study

Background: More contagious SARS-CoV-2 virus variants, breakthrough infections, waning immunity, and differential access to COVID-19 vaccines account for the worst yet numbers of hospitalization and deaths during the COVID-19 pandemic, particularly in resource-limited countries. There is an urgent need for clinically valuable, generalizable, and parsimonious triage tools assisting appropriate allocation of hospital resources during the pandemic. We aimed to develop and extensively validate a machine learning-based tool for accurately predicting the clinical outcome of hospitalized COVID-19 patients.Methods: CODOP was built using modified stable iterative variable selection and linear regression with lasso regularisation. To avoid generalization problems, CODOP was trained and tested with three time-sliced and geographically distinct cohorts encompassing 40 511 blood-based analyses of COVID-19 patients from more than 110 hospitals in Spain and the USA during 2020-21. We assessed the discriminative ability of the model using the Area Under the Receiving Operative Curve (AUROC) as well as horizon and Kaplan-Meier risk stratification analyses. To reckon the fluctuating pressure levels in hospitals through the pandemic, we offer two online CODOP calculators suited for undertriage or overtriage scenarios. We challenged their generalizability and clinical utility throughout an evaluation with datasets gathered in five hospitals from three Latin American countries. Findings: CODOP uses 12 clinical parameters commonly measured at hospital admission and associated with the pathophysiology of COVID-19. CODOP reaches high discriminative ability up to nine days before clinical resolution (AUROC: 0·90-0·96, 95% CI 0·879-0·970), it is well calibrated, and it enables an effective dynamic risk stratification during hospitalization. The two CODOP online calculators predicted the clinical outcome of the majority of patients (73-100% sensitivity and 84-100% specificity) from the distinctive Latin American evaluation cohort.Interpretation: The high predictive performance of CODOP in geographically disperse patient cohorts and the easiness-of-use, strongly suggest its clinical utility as a global triage tool, particularly in resource-limited countries.Funding: The Max Planck Society.Declaration of Interest: The authors declare no conflict of interest.Ethical Approval: This study was approved by the Provincial Research Ethics Committee of Málaga (Spain) and the Institutional Research Ethics Committees of each participating hospital.

Determination of parvovirus retention profiles in virus filter membranes using laser scanning microscopy

Virus filtration is a highly effective method in the downstream processing of biotherapeutic products to provide effective removal of potential infectious agents based on a size exclusion mechanism. The direct visualization of viruses retained inside the filter membrane represents a valuable tool to get a deeper understanding of the filtration process and to explain observations of virus breakthrough under particular operating conditions. Parvoviruses, which are used as worst-case models in validation studies, were purified and labeled with fluorescent dyes to detect their retention pattern inside the filter membrane using laser scanning microscopy. Critical factors influencing the reproducibility and accuracy of the approach were identified and optimized. The retention profiles revealed detectable differences between viruses, suggesting that the use of bacteriophages or nanoparticles as surrogates is limited in their applicability to accurately predict the behavior of parvoviruses in filter membranes. The established method enables a direct and quantitative analysis of the virus retention profile, adding a valuable tool to the conventional measurement of the viral load reduction to better understand the mechanism underlying the removal of viruses during nanofiltration of biotherapeutic products.

New insights into the performance characteristics of the Planova-series hollow-fiber parvovirus filters using confocal and electron microscopy.

Virus filtration remains a critical step in the downstream process for the production of monoclonal antibodies and other mammalian cell-derived biotherapeutics. Recent studies have shown large differences in virus capture behavior of different virus filters, although the origin of these differences is still unclear. The objective of this study was to use confocal and scanning electron microscopy to directly evaluate the capture of virus-size nanoparticles in Planova 20N and BioEX hollow-fiber virus filters. Confocal images of fluorescent nanoparticles were quantified using ImageJ image processing software based on the measured fluorescence intensity of the labeled nanoparticles. Nanoparticle capture by the Planova BioEX was independent of transmembrane pressure from 10 to 45 psi. In contrast, the Planova 20N showed significant differences in nanoparticle capture profile at low pressure, consistent with literature data showing virus breakthrough under these conditions. Images obtained after a process interruption show significant migration of previously captured nanoparticles in the Planova 20N filters but not in the BioEX. These results provide important insights into the nature of virus capture in different virus filters and its dependence on the underlying structure of the virus filtration membranes.

The HIV-1 reverse transcriptase E138A natural polymorphism decreases the genetic barrier to resistance to etravirine in vitro.

The HIV-1 reverse transcriptase (RT) natural polymorphism E138A is included among the mutations with a minor impact on response to etravirine. However, the interpretation of E138A on etravirine susceptibility is not consistent across different genotypic resistance algorithms. The aim of the study was to investigate the effect of E138A on the genetic barrier to resistance to etravirine in vitro. A panel of 20 clinically derived recombinant viruses (10 with WT 138E and 10 with 138A, all without any other resistance mutation) were cultured in the presence of increasing etravirine concentrations and analysed for genotypic changes at virus breakthrough. Parallel experiments were conducted with 138E/A/G/K/Q NL4-3-based clones. In the NL4-3 background, codon 138 changes increased etravirine resistance in the following order: Q > K > A > G > E. The 138A viruses were less susceptible to etravirine compared with the 138E viruses [median (IQR) fold change, 1.8 (1.5-2.8) versus 1.3 (0.8-1.8); P = 0.026], overcame etravirine pressure earlier [HR (95% CI) for viral outgrowth with 138A, 5.48 (2.95-28.24); P < 0.001] and grew at higher drug concentrations [median (IQR), 1350 (1350-1350) versus 0 (0-1350) nM; P = 0.005]. A variety of etravirine resistance-related mutations and changes in the RT connection and RNase H domains accumulated without any consistent pattern depending on baseline codon 138. E138A can contribute to reduced response to etravirine through a decreased genetic barrier to resistance. In vitro drug resistance selection is a valuable complement to define the full potential of low-level resistance mutations.

Transport and fate of viruses in sediment and stormwater from a Managed Aquifer Recharge site

Enteric viruses are one of the major concerns in water reclamation and reuse at Managed Aquifer Recharge (MAR) sites. In this study, the transport and fate of bacteriophages MS2, PRD1, and ΦX174 were studied in sediment and stormwater (SW) collected from a MAR site in Parafield, Australia. Column experiments were conducted using SW, stormwater in equilibrium with the aquifer sediment (EQ-SW), and two pore-water velocities (1 and 5 m day−1) to encompass expected behavior at the MAR site. The aquifer sediment removed >92.3% of these viruses under all of the considered MAR conditions. However, much greater virus removal (4.6 logs) occurred at the lower pore-water velocity and in EQ-SW that had a higher ionic strength and Ca2+ concentration. Virus removal was greatest for MS2, followed by PRD1, and then ΦX174 for a given physicochemical condition. The vast majority of the attached viruses were irreversibly attached or inactivated on the solid phase, and injection of Milli-Q water or beef extract at pH = 10 only mobilized a small fraction of attached viruses (<0.64%). Virus breakthrough curves (BTCs) were successfully simulated using an advective–dispersive model that accounted for rates of attachment (katt), detachment (kdet), irreversible attachment or solid phase inactivation (μs), and blocking. Existing MAR guidelines only consider the removal of viruses via liquid phase inactivation (μl). However, our results indicated that katt > μs > kdet > μl, and katt was several orders of magnitude greater than μl. Therefore, current microbial risk assessment methods in the MAR guideline may be overly conservative in some instances. Interestingly, virus BTCs exhibited blocking behavior and the calculated solid surface area that contributed to the attachment was very small. Additional research is therefore warranted to study the potential influence of blocking on virus transport and potential implications for MAR guidelines.

Overt and occult hepatitis B virus infection among community children in Northwest China.

Although a universal newborn hepatitis B (HB) immunization programme has been implemented in China, hepatitis B virus (HBV) breakthrough infection, including HB surface antigen (HBsAg)-positive infection and occult HBV infection (OBI), still occurs during infancy or childhood. Obtaining the actual prevalence of HBV infection in general children is important for preventing and controlling the spread of HB. Accordingly, we investigated the prevalence of overt infection and OBI in community children and compared the serological and virological characteristics of OBI and HBsAg carrier children to clarify the mechanisms related to OBI. In total, 6706 community children <12years of age were included from a population-based HBV seroepidemiological investigation in Northwest China. The HBsAg carrier rate in community children was 1.60% (107/6706), and the anti-HBs positive rate was 57.35% (3846/6706). Additionally, 1192 HBsAg-negative children were examined for OBI using nested PCR. The prevalence of OBI in local children was 1.26% (15/1192), and the predominant OBI genotypes were C and D. The 15 OBI children and 29 HBsAg-positive children from the same population did not have a statistical significant difference in age, gender, alanine aminotransferase (ALT), proportion of anti-HBs or anti-HBc, viral genotypes or mutations. Children with chronic overt infection had higher viral loads than OBI children (P=.004). These results suggested that HBV overt and occult infection of children was more serious in underdeveloped north-west regions. HBV neonatal immunization and catch-up programmes should be strengthened and supplemented. None of specific viral mutations or genotypes related to OBI were found. OBI may be a specific stage of HBV infection.

High resistance barrier to tenofovir alafenamide is driven by higher loading of tenofovir diphosphate into target cells compared to tenofovir disoproxil fumarate

Tenofovir alafenamide (TAF) is a new oral prodrug of tenofovir (TFV) recently approved for the treatment of HIV-1 as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. Clinical dosing with TAF vs. tenofovir disoproxil fumarate (TDF) has shown improved bone and kidney safety, and has been associated with an increased concentration of the anti-HIV active moiety tenofovir diphosphate (TFV-DP) in the PBMCs of treated patients and a reduction of TFV systemic exposure. We have studied the potential benefit of this increased concentration of TFV-DP observed clinically in an in vitro model system. Using a newly developed virus breakthrough assay with TAF exposure set at physiological concentrations, we show that HIV-1 clinical isolates harboring TFV resistance mutations such as K65R, 3 or 4 thymidine-analog mutations (TAMs), Q151M/K65R, or T69 insertion complex could be inhibited by TAF, but not by TFV when used at clinically relevant concentrations for TDF. These data suggest that the inhibitory quotient (IQ) of TAF is projected to be higher than the IQ of TDF, and that TAF has the potential to inhibit viruses containing TDF resistance in the clinic.

Off-treatment efficacy of 3-year nucleos(t)ide analogues in chronic hepatitis B patients

Lamivudine, telbivudine, and entecavir are the first-line drugs covered by the Taiwan National Health Insurance as 3-year treatments for patients with chronic hepatitis B virus (HBV), but the optimal treatment duration of each remains unclear. We aimed to detect HBV treatment-cessation durability, and compare the predictors in patients with and without clinical relapse. In this retrospective cohort study, 210 patients with chronic HBV who tested hepatitis B e-antigen positive or hepatitis B e-antigen negative were treated for 3 years with a nucleos(t)ide analogue. Of these, 102 patients continued therapy after 3 years, while 88 patients stopped treatment and were followed for 1 year due to financial difficulties. Efficacy was assessed in terms of alanine aminotransferase (ALT) level normalization, HBV DNA clearance, virus breakthrough, clinical relapse, and liver decompensation. The durability predictors were evaluated by host factors, HBV DNA, and drug differences. Eighty patients (14 on lamivudine, 19 on telbivudine, and 47 on entecavir) were recruited. There was no difference in clinical-relapse rate among lamivudine, telbivudine, and entecavir (35.7% vs. 36.8% vs. 31.9%, respectively; p = 0.916), and liver decompensated hepatitis was absent. In baseline clinical characteristics, there were no differences between the clinical-relapse and nonrelapse groups in age, sex, cirrhosis, prior treatment, HBV DNA, pretreatment ALT, or hepatitis B e-antigen (HBeAg). The mean 3rd year serum ALT level differed significantly between clinical-relapse and nonrelapse patients (37.5 U/L vs. 27.7 U/L, respectively; p = 0.044). The 3-year nucleos(t)ide analogue off-treatment in patients with chronic HBV delivered according to the Taiwan National Health Insurance guidelines had an overall 33.8% 1-year clinical-relapse rate without any decompensated hepatitis flare-ups.

Attenuation and colloidal mobilization of bacteriophages in natural sediments under anoxic as compared to oxic conditions

Redox conditions are known to affect the fate of viruses in porous media. Several studies report the relevance of colloid-facilitated virus transport in the subsurface, but detailed studies on the effect of anoxic conditions on virus retention in natural sediments are still missing. Therefore, we investigated the fate of viruses in natural flood plain sediments with different sesquioxide contents under anoxic conditions by considering sorption to the solid phase, sorption to mobilized colloids, and inactivation in the aqueous phase. Batch experiments were conducted under oxic and anoxic conditions at pH values between 5.1 and 7.6, using bacteriophages MS2 and PhiX174 as model viruses. In addition to free and colloid-associated bacteriophages, dissolved and colloidal concentrations of Fe, Al and organic C as well as dissolved Ca were determined. Results showed that regardless of redox conditions, bacteriophages did not adsorb to mobilized colloids, even under favourable charge conditions. Under anoxic conditions, attenuation of bacteriophages was dominated by sorption over inactivation, with MS2 showing a higher degree of sorption than PhiX174. Inactivation in water was low under anoxic conditions for both bacteriophages with about one log10 decrease in concentration during 16h. Increased Fe/Al concentrations and a low organic carbon content of the sediment led to enhanced bacteriophage removal under anoxic conditions. However, even in the presence of sufficient Fe/A-(hydr)oxides on the solid phase, bacteriophage sorption was low. We presume that organic matter may limit the potential retention of sesquioxides in anoxic sediments and should thus be considered for the risk assessment of virus breakthrough in the subsurface.